mycorrhizal research

8/18/2019 mycorrhizal research

1/19

CHAPTER4

H I S T O CH E M I CA L L O CA L I Z A T I O N O F

PO L Y PH O S PH A A PO L Y P)

GRANuLEs

A N D L I P I D B O D I E S I N T W O

A R B U S C U L A R M YC OR R S I Z A L F U N G I


2/19

INTRODUCTION

Plants and their AM fungal symbionts have coexisted for more than 400

million years (Redecker

et. al.

2000) and the association is widespread in terrestrial

ecosystems. Arbuscular mycorrhizal symbiosis provides multiple benefits for the

plants, not only enhanced P and nitrogen nutrition but also resistance against

pathogen and abiotic stresses (Smith

et al.

2003; Newsham

et al.

1995). Nutrition in

AM fungi is based on the acquisition of soil nutrients by the fungus (George

et al.

1995; Jakobsen, 1999) and fixation of atmospheric carbon by the plant (Ho and

Trappe, 1973) and on the exchange of these nutrients at specially adapted symbiotic

interfaces (Gianainezzi-Pearson

et al.

1991; Smith and R ead, 1997).

The enhanced growth rates of plants colonized by AM fungi are due to

improved mineral nutrition, particularly for P (Tinker, 1975). Nutrients taken up by

the fungus are transferred to the host and high P fluxes between 3.8x10

8 mo1 P

CM

-2 S 1

and 10 9 mol P cm

2 s 1

have been calculated and experimentally determined (Sanders

and Tinker, 1973; Pearson and Tinker, 1975). Such high rates of translocation require

mechanisms based upon bulk flow and cytoplasmic streaming (Tinker, 1975) and it

has been suggested that P may be translocated in a condensed form as polyP granules

by cyclosis Co x

et al. 1975).

Inorganic polyP is a linear polymer of phosphate linked by high-energy bonds

and a wide range of microorganisms store P by accumulation of poly P (Kornberg

et

106


3/19

al.

1999). It has been suggested that AM fungi accumulate poly P for long distance

translocation along hyphae (Cox

et al.

1980; Solaiman et al.

1999). High affinity

type Pi-transporter genes have been isolated from AM fungi, and gene products are

responsible for uptake of Pi from the soil solution and into the fungal cytosol

(Maldonodo-Mendoza

et al. 2001).

Recent attention has been given particularly to the mechanism of P uptake and

activity of selected enzymes in this symbiotic interaction. Callow

et al. 1978)

suggested that AM fungi synthesize polyP vacuolar granules from soil phosphate and

that the granules are broken down in the arbuscules to inorganic phosphate for release

to the host. Alkaline phosphatase activity specific to AM has been reported in Onion

and Tobacco (Bertheau, 1977). Gianiniazzi—Pearson and Gianiniazzi (1978)

determined that this activity is closely linked to both the mycorrhizal growth

stimulation and the arbuscular phase of colonization. They proposed that this enzyme

could play a role in the assimilation of P by mycorrhizal roots.

Arbuscular mycorrhizal fungi are oleogenic fungi and store large amount of

lipids as triacylglycerides (TAGs) (Gaspar

et al.

1994; Jagabaji-Hare, 1998). The

synthesis of TAG is a substantial sink for carbon in the intraradical hyphae (Pfeffer

et

al.

1999; Bago et al.

2000). Synthesis of lipids by the fungal endophyte has been

suggested as an alternate storage sink for the plant photosynthates (Cox

et al. 1975).

107


4/19

This chapter reports histochemical staining of polyphosphate granules and lipid

bodies in the roots of

Coleus

sp. inoculated with two AM fungal species.

MATERIALS AND METHODS

Plant and Fungus m aterial

Spores of two AM fungal species viz.,

Gigaspora albida

and

Gloms clarum

isolated from monospecific cultures using

Coleus

sp. as a host were used for the

study. Intact spores mounted in PVLG (Poly-vinyl Lacto Glycerol) (Koske and

Tessier, 1983) with or without Melzers reagent were examined under Leica

compound microscope, identified based on spore morphology, subcellular characters

and compared with original descriptions (Schenck and Perez, 1990; Almeida and

Schenck, 1990; Redecker

et al.

2000; Morton and Redecker, 2001; Schubler

et al.

2001). Spore morphology was also compared with the culture data established by

International Collection of Vesicular Arbuscular Fungi INVAM)

(http://invam.cag

,wvu .edu).

The inoculum (spores) was mixed with autoclaved soil sand at a ratio of 1:3

and filled in to 15 cm diameter pots. Pots were planted with

Coleus

cuttings and

watered regularly with sterile distilled water. Hoagland solution (Hoagland and

Arnon, 1938) was added every 15 days. After 40 days, watering was stopped and on

the 45

t day, roots of Coleus

sp. were checked for colonization using 0.5 trypan

blue (Koske and Gemma, 1989).

108


5/19

Histochemical assessment for PolyP an d Lipid droplets

_Coleus

root (45 days old) pieces were also stained for polyP granules using

Toluidine blue 0 (TBO) (Kumble and Kornberg, 1996), and Sudan Black for staining

lipid bodies (McGee-Russell and Smale, 1963).

Preparation of Toluidine blue :

1 g of Toluidine blue 0 was dissolved in

distilled water and 1N HC1 added to adjust the pH to 1. For polyP staining, roots

were gently washed in tap water and cut into lcm root pieces, cleared in 2 KOH at

90

C for 30-45min, thoroughly rinsed in water, acidified with 5N HC1 (Koske and

Gemmae, 1989), stained with Toluidine blue for 20 minutes and then rinsed with

distilled water.

Preparation of Sudan Black

For staining of lipid droplets, 100mg of Sudan

black in 10m1 of 70 saturated solution of ethyl alcohol was prepared and then

filtered before staining (Baker, 1960). Root pieces were stained in Sudan black for 1-

5 minutes after 1N HCl treatment (Koske and Gemma, 1989).

Photomicrographs of the stained root pieces under different magnifications

were taken using Olympus DP12-Olympus BX41 compound microscope. Staining

reactions were examined in the intraradical hyphae, arbuscules and vesicles of AM

fungi.

109


6/19


7/19

Plate VI Arbuscu lar colonization in a i g a s p o r a a l b i d a

a)

Early stage of arbuscule formation in roots of Coleus

sp. inoculated with

Gi. albida

(X 400).

b)

c) Dichotomously branched arbuscules (X 400).

d) D egenerating arbuscules (X 400).


8/19


9/19

Plate VIII Intraradical spores and Extraradical spores

a)

Extraradical spores of

Gloms clarum

in roots of

Coleus

sp. stained in

trypan blue (X 100).

b) Intraradical spores of

G . clarum unstained in roo ts X 400).

c)

Extraradical spore of Gigaspora albida

stained in trypan blue X10 0).

d)

Closer view of extraradical spore of Gi. albida X 400).


10/19

Plate VIII


11/19

of lipid bodies until it filled the entire cell to form a mature vesicle

(Plate VII b-f).

The matured vesicles later developed into intraradical spores by wall thickenings.

Histochemical observations revealed that intercellular hyphae contained polyP

granules that stained metachromatically with Toludine blue 0. Accumulation of

polyP granules was located in the intercellular hyphae in roots inoculated with

G .

albida

which were stained pinkish purple and there was a net transport of polyP

granules into the arbuscules by protoplasmic streaming

(Plate IX a-b).

In G. clarum

lipid bodies were present in the form of droplets of varying sizes

in the intercellular hyphae, stained blue black. Vesicles developing into intraradical

spores in the root cortex were also observed in the root, which stained blue-black.

Accumulation of lipid bodies in the spores through intercellular hyphae was observed

in the roots colonized by

G . clarum.

Some intracellular vesicles appeared to be almost

completely filled by a large lipid droplet. The movement of such lipid bodies may be

carried out possibly by cytoplasmic streaming. Vesicles formed were oval to elliptical

when formed intercellularly whose wall thicken to produce spores were also observed

in the roots of G. clarum

(Plate IX c-f).

DIS USSION

The presence of arbuscules in Gi. albida and vesicles and arbuscules in

G .

clarum

stained in trypan blue were recorded in the present study. Arbuscules are

111


12/19

Plate VII Vesicular colonization in l o m u s c l a r u m

a)

Lipid droplets in intercellular hyphae of

Coleus

sp. inoculated with

G .

clarum

X 400 ).

b)

Swelling (s) of intercellular hyphae (X 400).

c)

d) e) Movement of lipid bodies in intercellular hyphae (X 400).

0 Mature vesicle in the roots of

Coleus sp. X 400 ).


13/19

Plate VII


14/19

Plate IX Polyphosphate granules and lipid bodies

a) & b) Toludine blue 0 stained Polyphosphate granules in intercellular

hyphae of

Coleus

sp. inocu lated with

Gigaspora albida

X 400).

c) Sudan black stained lipid bodies in intercellular hyphae (X 400).

d), e & f) Sudan black stained intraradical spores of

Glarum clarum

X 400 ).


15/19

Plate X


16/19

thought to be the major sites of nutrient transfer. One of the major nutrients involved

is P and it is generally considered that the pathway of transfer is from soil to plant,

through the uptake by external mycelia, then into intercellular hyphae (in arum-type

mycorrhizas), arbuscular trunk hyphae via the arbuscule branches to the arbuscular

interface and then to the plant cell (Smith and Read, 1997). There have been studies

on arbuscule development using both light and electron microscopy. The

developmental cycle from the formation of the first arbuscule branches to final

collapse has been estimated to be between 4 and 12d in different plant/fungal

combinations (Toth and Miller, 1984; Alexander

et al.

1989). The activity of the

arbuscules at different stages of development has usually been assessed on the basis

of the physical appearance of the branches in electron microscopy or by light

microscopy after nonvital staining (with trypan blue). The electron microscopy

studies indicated the presence of cross walls within fine arbuscular branches (Kaspari,

1973; Bonfante-Fasolo et al.

1990). The presence of cross walls is highly correlated

with loss of activity of arbuscules and is often formed at sites between metabolically

active and inactive fungal regions. It has been reported that P could be transported to

the arbuscules when both intercellular hyphae and arbuscules are active and before

the formation of the crosswall (Dickson and Smith, 2001). High metabolic activity

was reported during early stage of arbuscule formation (Dickson and Smith, 2001).

Metachromatically stained polyP granules observed in the intercellular hyphae

of Gi. albida

have an essential role of transitory storage of P in the fungus vacuoles

before its transfer to the host plant. Cox

et al. (1975) observed cytochemical

112


17/19

identification of polyP granules in vacuoles within inter- and intra-cellular hyphae of

G

mosseae

Gerd & Trappe (Nicol. & Gerd.) in Onion roots. Ling Lee

et al. 1975)

also reported the occurrence of polyP granules in a wide range of ectotrophic and

endotrophic mycorrhizas. Polyphosphate granules occupy a total volume fraction

0.008 of the fungal structures within the root (Cox

et al.

1980).

Localization of polyP granules stained with Toludine blue 0 (TBO) has been

observed earlier in roots of Medicago truncatula

inoculated with G

versiforme

Javot

et al.

2007). Histochemical observations on AM fungi reported that hyphae contained

small granules which stain with lead and react metachromatically with Toluidine Blue

Cox

et al.

1975). These staining reactions suggest that the granules contain

polyphosphate in common with similar structures in other micro-organisms (Harold,

1966). These observations suggest that polyphosphates are of fundamental

importance to the phosphorus nutrition of mycorrhizal associations.

Inorganic P is translocated through the AM fungal hyphae as polyP granules

and after hydrolysis in the arbuscule, Pi is exported from the AM fungus to the

periarbuscular space (Ezawa

et al.

2003, Kohijima and Saito, 2004). Alternatively,

arbuscule death could be triggered by the plant either actively by acceleration of the

normal mechanism of arbuscular turnover or simply as a consequence of inadequate

carbon flow from the plant (Javot

et al.

2007). PolyP accumulated in the vacuolar

compartment can be translocated from the extraradical hyphae to intraradical hyphae

113


18/19

possibly via

cytoplasmic streaming or along a motile tubular vacuolar system (Uetake

et al.

2002). Once Pi has been released in the intraradical hyphae, it is assumed to be

transferred to the periarbuscular apoplastic compartment and from there it is available

to the plant. In the arbuscule, the polyphosphate may then be broken down by an

enzyme of polyphosphate kinase type (producing ATP) or polyphosphatase

(liberating Pi) (Harold, 1966). The products of break down may then be utilized in Pi

transfer across the arbuscule into the host. Alternatively, polyphosphate may serve in

phosphorylation mechanisms for the active transport of carbon skeleton into the

arbuscule from the host (Woolhouse, 1975) either through ATP produced by

degradative polyphosphate kinase action, or through a direct phosphorylation of

sugars by an enzyme of the polyphosphate glucokinase type (Szymona, 1962).

Results of the present study revealed the presence of lipid droplets in

intercellular hyphae and vesicles of

G. clarum.

Lipid staining of AM fungus

colonized roots has demonstrated abundant lipid in the vesicles and intercellular and

external hyphae (Cox

et al.

1975). Formation of spores from vesicles filled with

small lipid droplets, which later become a globule, was observed. Similar

observations were recorded earlier in spores of

Glomus

species (Maia and

Kimbrough, 1998). This supports the fact that AM fungi need a compact form of

carbon for translocation and storage, which could be the reason for abundant lipid

bodies (Bago

et al.

2002). The major fluxes of carbon in intraradical mycelium thus

appear to be efficient uptake of host derived hexose conversion to trehalose and

114


19/19

glycogen as storage forms and the synthesis of large amount of storage lipids. One of

the most crucial changes is the differentiation of the fungal partner in two

metabolically different but interconnected parts: the intraradical and the extra radical

mycelium, with the former being the only location for hexose acquisition. Such

hexose acquisition fuels metabolism and growth of intraradical mycelium, and it is

transferred into typical fungal molecules such as trehalose, glycogen and chitin. In

order to fuel extra radical fungal development, glycogen is translocated along newly

developed runner hyphae, which would extend the extraradical hyphal fungal colony

within the soil. These lipid bodies also play key roles in fungal morphogenic and

reproductive events such as sporulation (Bago

et al.

2002).

115

mycorrhizal research

Documents